The process of identifying a new drug candidate is long and tedious with many promising compounds eliminated from development during preclinical toxicity testing in animals. One reason for the high number of drop out compounds during the preclinical phase is the lack of useful toxicity data early in the discovery program. Many pharmaceutical companies have recognized this in the last 2 to 3 years [Cockerell et al., Toxicol. Path. vol 27, no. 4, p 471, (1999); Burkhardt et al., Toxicol. Path. vol 27, no. 4, p 472-473 (1999); Ryan, Toxicol. Path. vol 27, no. 4, p 474-476 (1999); Cockerell et al., Toxicol. Path. vol 27, no. 4, p 477-478, (1999)].
To date in vivo toxicity of a given candidate substance as a potential drug has involved the use of animal models. Underlying the animal tests is the assumption that the effects that agents produce in animals are applicable and predictive of effects in humans. In general, when the dosage is based on a per unit of body surface area, toxicology data from animals is applicable to humans. On the other hand, when the dosage is based on animal body weight, humans are more susceptible to toxicity than the test animals. Nevertheless, the vast majority of drugs are developed to be given on the basis of body weight.
Additionally, the actual numbers of animals used in drug testing are much lower than the human population likely to be exposed to the drug. For example, a 0.01% incidence of human exposure to a given drug means that approximately 25,000 out of 250 million individuals are exposed to a drug. To detect such a low incidence in animals would require that 30,000 animals be exposed to the drug. This is clearly an impractical number considering the variety of drugs in development at any given time. Consequently, exposure of fewer animals to high doses of candidate substances is desirable to identify hazards to humans exposed to low doses.
The use of animals in the initial stages of drug development is an expensive and inefficient method of producing toxicological data for new drugs especially in light of the fact that most chemicals this early in development will not be considered drug candidates. Various companies have tried to employ other methods of toxicological screening before the putative drugs enter the arena of animal testing.
A common approach to solving the toxicology data deficit has been to incorporate in vitro toxicity testing of promising new drugs into the drug discovery process at a time when new compounds are being identified for potency and efficacy against therapeutic targets. Quality toxicity data at this early stage permits pharmaceutical chemists to attempt to “design out” toxicity while maintaining efficacy/potency. Although this is a good idea in principle, in practice it has been extremely difficult to develop robust in vitro toxicity data and to match in vitro data with in vivo toxicity.
Key issues have been deciding on the type and nature of assays to be utilized and the test system to be employed. There are many biochemical and molecular assays that claim to assess toxicity in cells grown in culture. However, when only one or even two assays are used over a limited range of exposure concentrations, the probability of false negative and false positive data is high. Some of the most commonly used assays include, but are not limited to, leakage of intracellular markers as determined by lactate dehydrogenase (LDH), glutathione S-transferase (GST), and potassium, and the reduction of tetrazolium dyes such as MTT, XTT, Alamar Blue, and INT. All have been used as indicators of cell injury. In all cases, the screens typically only involve the use of one or two endpoints. The resulting data provides a yes/no or live/dead answer. This minimalist approach to the toxicity-screening problem has resulted in little progress towards developing a robust screening system capable of providing a useful toxicity profile that has meaning for predicting similar toxicity in animals. Therefore, there remains a need in the art for the development of new screening systems that provide more useful toxicity information, especially toxicity information that can be obtained rapidly and cost-effectively at early stages of the drug discovery process. A need exists for toxicity screening systems that do not require the use of animals but that provide reliable information on relative toxicity, mechanism of toxicity, and that effectively predict in vivo toxicity.